Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8 A hyperthermostable aldose-1-dehydrogenase with broad substrate specificity

被引:18
作者
Asada, Yukuhiko [2 ]
Endo, Satoshi [1 ,3 ]
Inoue, Yukari [1 ]
Mamiya, Hiroaki [1 ]
Hara, Akira [1 ]
Kunishima, Naoki [2 ]
Matsunaga, Toshiyuki [1 ]
机构
[1] Gifu Pharmaceut Univ, Biochem Lab, Gifu 5028585, Japan
[2] RIKEN, SPring Ctr 8, Harima Inst, Sayo, Hyogo 6795148, Japan
[3] Gifu Univ, United Grad Sch Drug Discovery & Med Informat Sci, Gifu 5011193, Japan
关键词
Short-chain dehydrogenase/reductase; Aldose; 1-dehydrogenase; Crystal structure; Thermostability; Aromatic-aromatic interaction; Thermus thermophilus; CRYSTAL-STRUCTURE; GLUCOSE-DEHYDROGENASE; EXTREME THERMOPHILE; ANGSTROM RESOLUTION; CARBONYL REDUCTASE; PATHWAY; COMPLEX; MOUSE; SDR; CRYSTALLOGRAPHY;
D O I
10.1016/j.cbi.2008.09.018
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80, C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD(+)-dependent aldose 1-dehydroganse, which accepts various aldoses such as D-fucose, D-galactose, D-glucose, L-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol. 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2-13 and up to 85 C. We have determined the crystal structure of the enzyme-NAD+ binary complex at 1.65 angstrom resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic-aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp-Gly-Gly sequence at positions 242-244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
引用
收藏
页码:117 / 126
页数:10
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